JP7481255B2 - Antibody-drug conjugate sensitivity markers - Google Patents

Description

The present invention relates to a method for identifying a subject to administer a pharmaceutical containing an antibody-drug conjugate to a human patient suffering from cancer.

Most anticancer drugs are effective in certain human patients but not in others. This is due to the genetic diversity of cancers and can even be observed between cancers in the same patient. The difference in efficacy between patients is particularly pronounced for molecular targeted anticancer drugs. Therefore, appropriate tests are needed to determine which anticancer drugs are effective for which patients, and without such tests, sufficient efficacy of anticancer drugs cannot be expected. The establishment of diagnostic methods based on the discovery of sensitivity markers is a means to accelerate the development of new anticancer drugs by identifying patients who are likely to show a clinical response to the drugs. This makes it possible to significantly reduce the scale, duration, and cost of clinical trials. Genomics, proteomics, or molecular imaging techniques should enable the rapid and sensitive detection of sensitivity markers. However, despite the availability of various techniques related to gene profiling in cancer, it is difficult to say that sensitivity markers for anticancer drugs are in the range of widespread practical use.

An example of a target for the above-mentioned molecular targeted anticancer drug is human TROP2. Human TROP2 (trophoblast cell surface protein 2, TACSTD2: tumor-associated calcium signal transducer 2, GA733-1, EGP-1, M1S1; hereafter referred to as hTROP2) is a type 1 cell membrane protein with a single transmembrane domain consisting of 323 amino acid residues.

Immunohistochemical analysis using clinical specimens has shown that hTROP2 is overexpressed in various epithelial cell-derived cancers, and that in normal tissues, expression is limited to epithelial cells of some tissues, and the expression level is lower than in tumor tissues (Non-Patent Documents 1-5). It has also been reported that hTROP2 expression correlates with poor prognosis in colorectal cancer (Non-Patent Document 1), gastric cancer (Non-Patent Document 2), pancreatic cancer (Non-Patent Document 3), oral cancer (Non-Patent Document 4), and glioma (Non-Patent Document 5). Furthermore, a model using colon cancer cells has reported that hTROP2 expression is involved in anchorage-independent cell growth of cancer cells and tumor formation in immunodeficient mice (Non-Patent Document 6).

Based on information suggesting such a relationship with cancer, several anti-hTROP2 antibodies have been established and their antitumor effects have been investigated. Among these, antibodies that show antitumor activity in nu/nu mouse xenograft models using only the antibody (Patent Documents 1 to 4) as well as antibodies that show antitumor activity as antibody-drug conjugates (Patent Documents 5 to 7) have been disclosed. However, the strength and scope of their activity are still insufficient, and there is an unmet medical need for therapeutically targeting hTROP2. The reasons why existing antibodies or antibody-drug conjugates do not meet the medical needs include insufficient efficacy as medicines and the fact that appropriate sensitivity markers have not been found. For example, it is known that the antitumor activity of antibody-drug conjugates targeting hTROP2 in small cell lung cancer cannot be predicted by the expression level of hTROP2 (Non-Patent Document 7).

Antibody-drug conjugates (hereinafter sometimes referred to as "ADCs"), which are antibodies that bind to antigens that are expressed on the surface of cancer cells and can be internalized into the cells and have a cytotoxic drug bound thereto, are expected to selectively deliver drugs to cancer cells, thereby accumulating the drugs in the cancer cells and killing the cancer cells. As one of such antibody-drug conjugates, an antibody-drug conjugate containing an antibody and a derivative of exatecan, a topoisomerase I inhibitor, as components, is known (Patent Documents 9 to 15, Non-Patent Documents 8 to 16). The antibody-drug conjugate described in Patent Document 9 contains an anti-hTROP2 antibody and can kill cancer cells expressing hTROP 2 , but as with existing antibodies or antibody-drug conjugates that target hTROP2, there is a possibility that antitumor activity cannot be accurately predicted only by the expression level of hTROP2.

Human SLFN11 (Schlafen family member 11) is a protein consisting of 901 amino acid residues, and it has been suggested that it binds to replication forks in response to DNA replication stress and inhibits DNA replication (Non-Patent Document 17). It has also been reported that the sensitivity of cancer cell lines to DNA-damaging anticancer drugs, including topoisomerase I inhibitors, is highly correlated with the mRNA expression level of SLFN11 (Non-Patent Documents 18-19). It is also known that the combined use of veliparib, a poly ADP-ribose polymerase (PARP) inhibitor, and rovalpituzumab tesirine (Rova-T), an anti-DLL3 antibody-drug conjugate, has a survival benefit in small cell lung cancer patients who highly express SLFN11 (Non-Patent Document 20). Furthermore, it is known that the combined use of alkylating agents Temozolamide and Veliparib has a life-prolonging effect on small cell lung cancer patients who highly express SLFN11 (Non-Patent Document 21). However, the relationship between the antitumor activity of ADCs using topoisomerase I inhibitors such as exatecan and the expression level of SLFN11 has not yet been clarified, and its effectiveness as a diagnostic agent for predicting drug efficacy is also unknown.

International Publication No. WO 2008/144891 International Publication No. 2011/145744 International Publication No. 2011/155579 International Publication No. 2013/077458 International Publication No. 2003/074566 International Publication No. 2011/068845 International Publication No. 2013/068946 U.S. Pat. No. 7,999,083 International Publication No. 2014/057687 International Publication No. 2014/061277 International Publication No. 2015/098099 International Publication No. 2015/115091 International Publication No. 2015/146132 International Publication No. 2015/155976 International Publication No. 2015/155998

Ohmachi T, et al., Clin. Cancer Res., 12(10), 3057-3063 (2006). Muhlmann G, et al., J. Clin. Pathol., 62(2), 152-158 (2009). Fong D, et al., Br. J. Cancer, 99(8), 1290-1295 (2008). Fong D, et al., Mod. Pathol., 21(2), 186-191 (2008). Ning S, et al., Neurol. Sci., 34(10), 1745-1750 (2013). Wang J, et al., Mol. Cancer Ther., 7(2), 280-285 (2008). Gray J. E., et al. Clin. Cancer Res. 23(19), 5711-5719 (2017) Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. Alley, S. C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537. Damle N. K. Expert Opin. Biol. Ther. (2004) 4, 1445-1452. Senter P. D., et al., Nature Biotechnology (2012) 30, 631-637. Howard A. et al., J Clin Oncol 29: 398-405. Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108. Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046. Doi T, et al., Lancet Oncol 2017;18:1512-22. Takegawa N, et al., Int. J. Cancer: 141, 1682-1689 (2017) Murai J, et al., Mol. Cell 69(3), 371-384 (2018) Zoppoli G, et al., Proc. Natl. Acad. Sci. U.S.A., 109(37):15030-15035 (2012) Barretina J, et al., Nature 483(7391), 603-607 (2012) Van Den Borg R, et al., Expert Rev. Anticancer Ther., 19 (6), 461-471 (2019) Pietanza MC, et al., J. Clin. Oncol. 36 (23), 2386-2394 (2018)

The present invention relates to a method for identifying subjects for administration of a medicine containing an anti-hTROP2 antibody to a human patient suffering from cancer, using the amount of gene expression at the mRNA level as an indicator.

The inventors discovered that by combining the expression levels of the mRNA of the hTROP2 gene and the SLFN11 gene, it is possible to more accurately identify subjects to be administered a pharmaceutical containing an anti-hTROP2 antibody, and thus completed the present invention.

That is, the present invention includes, but is not limited to, the following items.
[1] A method for identifying a human patient suffering from cancer to whom a pharmaceutical agent containing an anti-hTROP2 antibody is to be administered, comprising:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the hTROP2 gene at the mRNA level in the biological sample;
3) evaluating the expression level of the SLFN11 gene at the mRNA level in the biological sample in which the expression level of the hTROP2 gene has been determined to be high; and 4) identifying a human patient having the biological sample in which the expression level of the SLFN11 gene has been determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[2] A method for identifying a human patient suffering from cancer to whom a pharmaceutical agent containing an anti-hTROP2 antibody is to be administered, comprising:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression levels of the hTROP2 gene and the SLFN11 gene at the mRNA level in the sample; and 3) identifying a human patient having the sample in which the expression levels of the hTROP2 gene and the SLFN11 gene are determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[3] The method according to [1] or [2], in which the log ₂ [RPKM+1] value is measured by RNA sequencing from a biological sample obtained from a human patient diagnosed with cancer, and if this value exceeds a specific value, the expression level of the hTROP2 gene and/or the SLFN11 gene at the mRNA level is determined to be high.
[4] The method according to [3], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the _log2 [RPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0.
[5] The method according to [3] or [4], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the _log2 [RPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[6] The method according to any one of [3] to [5], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the _log2 [RPKM+1] value is greater than any one selected from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.
[7] The method according to any one of [3] to [6], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than any one selected from the group consisting of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.
[8] The method according to any one of [3] to [7], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than any one selected from the group consisting of 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.
[9] The method according to any one of [3] to [7], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value exceeds any one selected from the group consisting of 7.0, 7.5, and 8.0.
[10] The method according to [9], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than 7.0.

[11] The method according to [9], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than 7.5.
[12] The method according to [9], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than 8.0.
[13] The method according to any one of [3] to [12], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the _log2 [RPKM+1] value is greater than any one selected from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0.
[14] The method according to any one of [3] to [13], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the _log2 [RPKM+1] value is greater than any one selected from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.
[15] The method according to any one of [3] to [14], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the _log2 [RPKM+1] value exceeds any one selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.

[16] The method according to any one of [3] to [14], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value exceeds any one selected from the group consisting of 1.0, 2.0 and 3.0.
[17] The method according to [16], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than 1.0.
[18] The method according to [16], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than 2.0.
[19] The method according to [16], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [RPKM+1] value is greater than 3.0.
[20] The method according to [1] or [2], in which the log ₂ [FPKM+1] value is measured by RNA sequencing from a biological sample obtained from a human patient diagnosed with cancer, and if this value exceeds a specific value, the expression level of the hTROP2 gene and/or the SLFN11 gene at the mRNA level is determined to be high.

[21] The method according to [20], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.
[22] The method according to [20] or [21], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value exceeds any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0.
[23] The method according to any one of [20] to [22] , wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 6.0 or 7.0.
[24] The method according to [ 23 ], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 6.0.
[25] The method according to [ 23 ], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 7.0.

[26] The method according to any one of [20] to [25], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than any one selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0.
[27] The method according to any one of [20] to [26], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value exceeds any one selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.
[28] The method according to any one of [ 20 ] to [ 27 ], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 2.0 or 3.0.
[29] The method according to [28], wherein the expression level of the SLFN11 gene at the mRNA level is judged to be high when the log2[FPKM+1] value is greater than 2.0.
[30] The method according to [28], wherein the expression level of the SLFN11 gene at the mRNA level is judged to be high when the log2[FPKM+1] value is greater than 3.0.

[31] The method according to [1] or [2], wherein the log2[MNC+1] value is measured by EdgeSeq Assay from a biological sample obtained from a human patient diagnosed with cancer, and if the log2[MNC+1] value exceeds a specific value, the expression level of the hTROP2 gene and/or the SLFN11 gene at the mRNA level is determined to be high.
[32] The method according to [31], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than any one selected from the group consisting of 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, and 15.0.
[33] The method according to [31] or [32], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than any one selected from the group consisting of 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, and 14.0.
[34] The method according to any one of [31] to [33], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 12.0, 13.0, or 14.0 .
[35] The method according to [34], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 12.0.

[36] The method according to [34], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [MNC+1] value is greater than 13.0.
[37] The method according to [34], wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log ₂ [MNC+1] value is greater than 14.0.
[38] The method according to any one of [31] to [37], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the _log2 [MNC+1] value is greater than any one selected from the group consisting of 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, and 13.5.
[39] The method according to any one of [31] to [38], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the _log2 [MNC+1] value is greater than any one selected from the group consisting of 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, and 12.5.
[40] The method according to any one of [31] to [39], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the _log2 [MNC+1] value is greater than any one selected from the group consisting of 11.5, 12.0, and 12.5 .

[41] The method according to [40], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [MNC+1] value is greater than 11.5.
[42] The method according to [40], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [MNC+1] value is greater than 12.0.
[43] The method according to [40], wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log ₂ [MNC+1] value is greater than 12.5.
[44] The method according to any one of [1] to [43], wherein the biological sample comprises a tumor sample.
[45] The method according to any one of [1] to [44], wherein the medicine containing an anti-hTROP2 antibody is an anti-hTROP2 antibody-drug conjugate.

[46] The anti-hTROP2 antibody-drug conjugate has the following formula:

(In the formula, A represents the binding site to the anti-hTROP2 antibody.)
and the anti-hTROP2 antibody are linked via a thioether bond.
[47] The method according to [46], wherein the anti-hTROP2 antibody is an antibody having a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 2.
[48] The method according to [47], wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-hTROP2 antibody is deleted.
[49] The method according to any one of [46] to [48], wherein the average number of drug-linker structures bound per antibody is in the range of 2 to 8.
[50] The method according to any one of [46] to [49], wherein the average number of drug-linker structures bound per antibody is in the range of 3.5 to 4.5.

[51] The method according to [45], wherein the anti-hTROP2 antibody-drug conjugate is Sacituzumab Govitecan (IMMU-132).
[52] The method according to any one of [1] to [51], wherein the cancer is lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, biliary tract cancer, thyroid cancer, lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, and/or multiple myeloma.

Furthermore, the present invention also includes the following items. Note that the elements or requirements of [3] to [52] can also be applied to the following inventions.

[53] A method for identifying a subject to be administered a medicament containing an anti-hTROP2 antibody in a human patient suffering from cancer, comprising:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression levels of the hTROP2 gene and/or the SLFN11 gene at the mRNA level in the sample; and 3) identifying a human patient having the sample in which the expression levels of the hTROP2 gene and/or the SLFN11 gene are determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[54] A method for identifying a subject to be administered a medicament containing an anti-hTROP2 antibody in a human patient suffering from cancer, comprising:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the hTROP2 gene at the mRNA level in the sample; and 3) identifying a human patient having the sample in which the expression level of the hTROP2 gene is determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[55] A method for identifying a human patient suffering from cancer to whom a medicament containing an anti-hTROP2 antibody is to be administered, comprising: 1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the SLFN11 gene at the mRNA level in the sample; and 3) identifying a human patient having the sample in which the expression level of the SLFN11 gene is determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:

[56] A method for identifying a human patient suffering from cancer to whom a medicament containing an anti-hTROP2 antibody is to be administered, comprising: 1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression levels of the hTROP2 gene and the SLFN11 gene at the mRNA level in the sample; and 3) identifying a human patient having the sample in which the expression levels of the hTROP2 gene and the SLFN11 gene are determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[57] A method for identifying a human patient suffering from cancer to whom a medicament containing an anti-hTROP2 antibody is to be administered, comprising: 1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the hTROP2 gene at the mRNA level in the sample;
3) evaluating the expression level of the SLFN11 gene at the mRNA level in the biological sample in which the expression level of the hTROP2 gene has been determined to be high; and 4) identifying a human patient having the biological sample in which the expression level of the SLFN11 gene has been determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[58] A method for identifying a human patient suffering from cancer to whom a pharmaceutical agent containing an anti-hTROP2 antibody is to be administered, comprising: 1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the SLFN11 gene at the mRNA level in the sample;
3) evaluating the expression level of the hTROP2 gene at the mRNA level in the biological sample in which the expression level of the SLFN11 gene has been determined to be high; and 4) identifying a human patient having the biological sample in which the expression level of the hTROP2 gene has been determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[59] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) assessing the expression level of the hTROP2 gene at the mRNA level in the biological sample;
3) evaluating the expression level of the SLFN11 gene at the mRNA level in the biological sample in which the expression level of the hTROP2 gene has been determined to be high; and 4) selecting a human patient having the biological sample in which the expression level of the SLFN11 gene has been determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[60] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression levels of the hTROP2 gene and the SLFN11 gene at the mRNA level in the sample; and 3) selecting a human patient having the sample in which the expression levels of the hTROP2 gene and the SLFN11 gene are determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:

[61] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression levels of the hTROP2 gene and/or the SLFN11 gene at the mRNA level in the sample; and 3) selecting human patients having the sample in which the expression levels of the hTROP2 gene and/or the SLFN11 gene are determined to be high, as subjects to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[62] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the hTROP2 gene at the mRNA level in the sample; and 3) selecting a human patient having the sample in which the expression level of the hTROP2 gene is determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody.
The method includes:
[63] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression level of the SLFN11 gene at the mRNA level in the sample; and 3) selecting a human patient having the sample in which the expression level of the SLFN11 gene is determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[64] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) evaluating the expression levels of the hTROP2 gene and the SLFN11 gene at the mRNA level in the sample; and 3) selecting a human patient having the sample in which the expression levels of the hTROP2 gene and the SLFN11 gene are determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes:
[65] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) assessing the expression level of the hTROP2 gene at the mRNA level in the sample;
3) evaluating the expression level of the SLFN11 gene at the mRNA level in the biological sample in which the expression level of the hTROP2 gene has been determined to be high; and 4) selecting a human patient having the biological sample in which the expression level of the SLFN11 gene has been determined to be high, as a subject to which a pharmaceutical agent containing an anti-hTROP2 antibody is to be administered;
The method includes:

[66] A method for treating cancer, comprising administering a medicament containing an anti-hTROP2 antibody:
1) obtaining a biological sample from a human patient diagnosed with cancer;
2) assessing the expression level of the SLFN11 gene at the mRNA level in the sample;
3) evaluating the expression level of the hTROP2 gene at the mRNA level in the biological sample in which the expression level of the SLFN11 gene has been determined to be high; and 4) selecting a human patient having the biological sample in which the expression level of the hTROP2 gene has been determined to be high, as a subject to which a pharmaceutical agent containing an anti-hTROP2 antibody is to be administered;
The method includes:

Effect of the Invention

By identifying the subjects to whom a drug containing an anti-hTROP2 antibody will be administered, it is possible to select patients who are likely to benefit from the drug and administer the drug to them.

The amino acid sequence of the humanized anti-hTROP2 antibody heavy chain (SEQ ID NO:1) is shown. The amino acid sequence of the humanized anti-hTROP2 antibody light chain (SEQ ID NO:2) is shown. The CDRH1 sequence (SEQ ID NO: 3), CDRH2 sequence (SEQ ID NO: 4), and CDRH3 sequence (SEQ ID NO: 5) of the humanized anti-hTROP2 antibody heavy chain, and the CDRL1 sequence (SEQ ID NO: 6), CDRL2 sequence (SEQ ID NO: 7), and CDRL3 sequence (SEQ ID NO: 8) of the humanized anti-hTROP2 antibody light chain are shown. FIG. 1 shows the cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in FaDu cells when SLFN11 was knocked down. FIG. 1 shows the cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in NCI-H1781 cells when SLFN11 was knocked down. FIG. 1 shows the cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in Calu-3 cells when SLFN11 was knocked down. FIG. 1 shows the cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in MDA-MB-468 cells when SLFN11 was knocked down. FIG. 1 shows the cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in HCC38 cells when SLFN11 was knocked down.

DEFINITIONS In this specification, unless otherwise stated, when referring to a numerical value, "about" means ±10% of the stated numerical value.

In this specification, "cancer" and "tumor" are used interchangeably.

As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA, and cRNA.

As used herein, the term "polynucleotide" is used synonymously with nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers.

In this specification, the terms "polypeptide" and "protein" are used interchangeably.

In this specification, "cells" includes cells within an animal body and cultured cells.

As used herein, "hTROP2" refers to the human protein encoded by the gene identified by accession number NM_002353 (NCBI) and allelic variants thereof, including the protein identified by accession number NP_002344 (NCBI).

As used herein, "SLFN11" refers to the human protein encoded by the gene identified by accession number NM_152270 (NCBI) and allelic variants thereof, including the protein identified by accession number NP_689483 (NCBI).

In the present specification, the term "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody that has binding activity to an antigen, and includes Fab, F(ab')2, Fv, scFv, diabody, linear antibody, and multispecific antibody formed from antibody fragments. Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions, is also included in the antigen-binding fragment of an antibody. However, it is not limited to these molecules as long as it has the ability to bind to an antigen. In addition, these antigen-binding fragments include not only full-length antibody protein molecules treated with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

In this specification, "CDR" means a complementarity determining region (CDR). It is known that there are three CDRs in each of the heavy and light chains of an antibody molecule. CDRs are also called hypervariable domains, and are located in the variable regions of the heavy and light chains of an antibody, and are sites with particularly high variability in the primary structure, and are separated into three sites in the primary structure of the heavy and light chain polypeptide chains. In this specification, the CDRs of the heavy chain are represented as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain are represented as CDRL1, CDRL2, and CDRL3 from the amino terminal side of the light chain amino acid sequence. These sites are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

As used herein, "response" to treatment means, with respect to a treated tumor, that the tumor exhibits (a) a slowing of growth, (b) cessation of growth, or (c) regression.

Anti-hTROP2 Antibody The antibody against hTROP2 used in the present invention can be obtained by immunizing an animal with hTROP2 or any polypeptide selected from the amino acid sequence of hTROP2, and collecting and purifying the antibody produced in the body, using a method commonly used in this field. The species of TROP2 that serves as an antigen is not limited to humans, and animals can also be immunized with TROP2 derived from animals other than humans, such as mice and rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibody that binds to a heterologous TROP2 with hTROP2.

In addition, according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)), a hybridoma can be established by fusing an antibody-producing cell that produces an antibody against hTROP2 with a myeloma cell, thereby obtaining a monoclonal antibody.

The antigen hTROP2 can be obtained by expressing the hTROP2 gene in a host cell through genetic manipulation. Specifically, a vector capable of expressing the hTROP2 gene is prepared, introduced into a host cell to express the gene, and the expressed TROP2 is purified. It is also possible to use the above-mentioned genetically manipulated hTROP2-expressing cells or a cell line expressing hTROP2 as the hTROP2 protein.

The antibodies of the present invention include not only the monoclonal antibodies against hTROP2, but also genetically engineered antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity against humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods.

An example of a humanized antibody is a humanized antibody consisting of a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2, but is not limited to this.

For example, various anti-hTROP2 antibodies described in WO 2008/144891, WO 2011/145744, WO 2011/155579, WO 2013/077458, WO 2003/074566, WO 2011/068845, WO 2013/068946, U.S. Patent No. 7,999,083, or WO 2015/098099 can be used in the present invention.

It is known that antibodies produced in cultured mammalian cells have deleted lysine residues at the carboxyl termini of their heavy chains (Tsubaki et al., Int. J. Biol. Macromol, 139-147, 2013). However, this deletion of the heavy chain sequence does not affect the antigen-binding ability or effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, in the present invention, antibodies in which the above-mentioned lysine residues at the carboxyl termini of the heavy chains are deleted can also be used.

The anti-hTROP2 antibody used in the present invention also includes an antigen-binding fragment of the antibody. Examples of the antigen-binding fragment of the antibody include Fab, F(ab')2, Fv, or single-chain Fv (scFv) in which heavy and light chain Fvs are linked with an appropriate linker, diabodies (diabodies), linear antibodies, and multispecific antibodies formed from antibody fragments. In addition, Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions, is also included in the antibody fragment.

The anti-hTROP2 antibody used in the present invention also includes modified antibodies. The modified antibody means an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include the attachment of a chemical moiety to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, and the like. Biological modifications include those that have been post-translationally modified (e.g., glycosylation to the N- or O-linkage, processing of the N- or C-terminus, deamidation, isomerization of aspartic acid, oxidation of methionine), and those that have been expressed using a prokaryotic host cell to add a methionine residue to the N-terminus. The anti-hTROP2 antibody used in the present invention also includes those that have been labeled to enable detection or isolation of the anti-hTROP2 antibody or hTROP2, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled modifications. Such modified anti-hTROP2 antibodies are useful for improving the stability and blood retention of the antibodies, reducing antigenicity, detecting or isolating anti-hTROP2 antibodies or hTROP2, and the like.

In addition, antibody-dependent cellular cytotoxicity can be enhanced by adjusting the sugar chain modification (glycosylation, defucosylation, etc.) attached to the anti-hTROP2 antibody used in the present invention. Known techniques for adjusting the sugar chain modification of antibodies include, but are not limited to, those disclosed in International Publication Nos. 1999/54342, 2000/61739, and 2002/31140. The anti-hTROP2 antibody used in the present invention also includes antibodies in which the sugar chain modification has been adjusted.

Other Antibodies The method of the present invention can also be used for pharmaceuticals containing antibodies that bind to antigens other than anti-hTROP2 antibodies. There are no particular limitations on the antibodies other than anti-hTROP2 antibodies used in the present invention, and examples of such antibodies include anti-HER2 antibodies, anti-HER3 antibodies, anti-B7-H3 antibodies, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD56 antibodies, anti-CD98 antibodies, anti-DR5 antibodies, anti-EGFR antibodies, anti-EPHA2 antibodies, anti-FGFR2 antibodies, anti-FGFR4 antibodies, anti-FOLR1 antibodies, anti-VEGF antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD70 antibodies, anti-PSMA antibodies, anti-CEA antibodies, and anti-M Examples of the antibody include esothelin antibody, anti-A33 antibody, anti-CanAg antibody, anti-Cripto antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-integrin antibody, anti-tenascin-C antibody, anti-SLC44A4 antibody, anti-GPR20 antibody, and anti-CDH6 antibody, and preferably include anti-HER2 antibody, anti-HER3 antibody, anti-B7-H3 antibody, anti-GPR20 antibody, and anti-CDH6 antibody, and more preferably include anti-HER2 antibody. Each antibody can be obtained by the same method as the anti-hTROP2 antibody. Furthermore, each antibody has universal properties that antibodies usually have, similar to the anti-hTROP2 antibody.

In the present invention, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (Human Epidermal Growth Factor Receptor Type 2; ErbB-2), and preferably has the activity of being internalized into HER2-expressing cells by binding to HER2. Examples of anti-HER2 antibodies include trastuzumab (U.S. Pat. No. 5,821,337) and pertuzumab (International Publication No. WO 01/00245), and preferably trastuzumab. In the present invention, the term "anti-HER3 antibody" refers to an antibody that specifically binds to HER3 (Human Epidermal Growth Factor Receptor Type 3; ErbB-3), and preferably has the activity of being internalized into HER3-expressing cells through binding to HER3. Examples of anti-HER3 antibodies include Patritumab (U3-1287), U1-59 (WO 2007/077028), MM-121 (Seribantumab), the anti-ERBB3 antibodies described in WO 2008/100624, RG-7116 (Lumretuzumab), and LJM-716 (Elgemtumab), and preferred examples include Patritumab and U1-59.

In the present invention, the term "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B cell antigen #7 homolog 3; PD-L3; CD276), and preferably has the activity of being internalized into B7-H3-expressing cells by binding to B7-H3. An example of an anti-B7-H3 antibody is M30-H1-L4 (WO 2014/057687).

In the present invention, the term "anti-GPR20 antibody" refers to an antibody that specifically binds to GPR20 (G protein-coupled receptor 20) and preferably has the activity of being internalized into GPR20-expressing cells by binding to GPR20. An example of an anti-GPR20 antibody is h046-H4e/L7 (WO 2018/135501).

In the present invention, the term "anti-CDH6 antibody" refers to an antibody that specifically binds to CDH6 (Cadherin-6), and preferably has the activity of being internalized into CDH6-expressing cells by binding to CDH6. An example of an anti-CDH6 antibody is H01L02 (WO 2018/212136).

The antibody-drug conjugate used in the present invention has the formula

(In the formula, A represents the binding site with the antibody.)
and an antibody via a thioether bond.

In the present invention, the partial structure of the antibody-drug conjugate consisting of a linker and a drug is referred to as a "drug linker." This drug linker is bonded to a thiol group (in other words, the sulfur atom of a cysteine residue) generated at the disulfide bond site between the antibody chains (two sites between the heavy chains and the heavy chains, and two sites between the heavy chains and the light chains).

The drug linker of the present invention is composed of the topoisomerase I inhibitor exatecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione)). Exatecan has the formula

It is a camptothecin derivative having antitumor effects, represented by the formula:

The antibody-drug conjugate used in the present invention may also be represented by the following formula:

Here, the drug linker is bound to the antibody via a thioether bond. In addition, n is synonymous with the so-called average drug binding number (DAR; Drug-to-Antibody Ratio) and indicates the average number of drug linkers bound per antibody. The average number of drug linkers bound per antibody in the antibody-drug conjugate used in the present invention can be adjusted in the range of 0 to 8, but is preferably 2 to 8. In the case of an anti-hTROP2 antibody, the average number of bonds is more preferably 3 to 5, and even more preferably 3.5 to 4.5.

The antibody-drug conjugate used in the present invention is cleaved from the linker after being transferred into the cancer cell, and the antibody-drug conjugate is

to release the compound represented by the formula:

The above compound is considered to be the main component of the antitumor activity of the antibody-drug conjugate used in the present invention, and has been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15; 22(20): 5097-5108, Epub 2016 Mar 29). If the antibody-drug conjugate releases the above compound, the method of the present invention can be applied without limiting the antigen recognized by the antibody to hTROP2.

Topoisomerase I is an enzyme involved in DNA synthesis, which changes the higher-order structure of DNA by breaking and recombining single strands of DNA. Therefore, drugs with topoisomerase I inhibitory activity can inhibit DNA synthesis, thereby stopping cell division at the S phase of the cell cycle (DNA synthesis phase) and inducing cell death by apoptosis, thereby suppressing the proliferation of cancer cells.

It is also known that the antibody-drug conjugate used in the present invention has a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). This bystander effect is exerted when the antibody-drug conjugate used in the present invention is internalized in a target-expressing cancer cell, and the compound exerts an antitumor effect on nearby cancer cells that do not express the target.

The drug linker intermediate used in the preparation of the antibody-drug conjugate used in the present invention is represented by the following formula:

The above drug linker intermediate can be expressed by the chemical name N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide, and can be produced with reference to the descriptions in International Publication No. WO 2015/098099 and the like.

The antibody-drug conjugate used in the present invention can be prepared by reacting the aforementioned drug linker intermediate with an antibody having a thiol group (also known as a sulfhydryl group).

Antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) can be used in an amount of 0.3 to 3 molar equivalents per antibody interchain disulfide, and reacted with the antibody in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), thereby obtaining an antibody having sulfhydryl groups in which the antibody interchain disulfides have been partially or completely reduced.

Additionally, 2 to 20 molar equivalents of drug linker intermediate can be used per antibody having a sulfhydryl group to produce antibody-drug conjugates in which 2 to 8 drugs are conjugated per antibody.

The average number of drugs bound per antibody molecule in the produced antibody-drug conjugate can be calculated, for example, by measuring the UV absorbance of the antibody-drug conjugate and its conjugation precursor at two wavelengths, 280 nm and 370 nm (UV method), or by treating the antibody-drug conjugate with a reducing agent, quantifying each of the resulting fragments by HPLC measurement, and then calculating the average number of drugs bound per antibody molecule (HPLC method).

Conjugation of the antibody and the drug linker intermediate, and calculation of the average number of drugs bound per antibody molecule in the antibody-drug complex can be performed with reference to the descriptions in WO 2015/098099, WO 2017/002776, etc.

The anti-hTROP2 antibody-drug conjugate having the above-mentioned drug linker used in the present invention may be an antibody-drug conjugate described in International Publication No. 2015/098099. The preferred anti-hTROP2 antibody-drug conjugate described in the International Publication contains an antibody having a heavy chain amino acid sequence containing CDRH1 (TAGMQ) consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, CDRH2 (WINTHSGVPKYAEDFKG) consisting of the amino acid sequence shown in SEQ ID NO: 4, and CDRH3 (SGFGSSYWYFDV) consisting of the amino acid sequence shown in SEQ ID NO: 5 in the heavy chain variable region, and a light chain amino acid sequence containing CDRL1 (KASQDVSTAVA) consisting of the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing, CDRL2 (SASYRYT) consisting of the amino acid sequence shown in SEQ ID NO: 7, and CDRL3 (QQHYITPLT) consisting of the amino acid sequence shown in SEQ ID NO: 8 in the light chain variable region. More preferred among the anti-hTROP2 antibody-drug conjugates described in the International Publication are those that contain an antibody consisting of a heavy chain amino acid sequence containing a heavy chain variable region consisting of the 20th to 140th amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, and a light chain amino acid sequence containing a light chain variable region consisting of the 21st to 129th amino acid residues of the amino acid sequence shown in SEQ ID NO: 2. Particularly preferred among the anti-hTROP2 antibody-drug conjugates described in the International Publication are those that contain an antibody consisting of the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2.

It is known that antibodies produced in cultured mammalian cells lack the lysine residue at the carboxyl terminus of their heavy chains. Therefore, the above antibody-drug conjugate also contains an antibody in which the lysine residue at the carboxyl terminus of the heavy chain is missing.

However, as long as the antibody contained therein can recognize hTROP2, the anti-hTROP2 antibody-drug conjugate used in the present invention is not limited to those having the above-mentioned specific drug linker. An example of such an anti-hTROP2 antibody-drug conjugate can be Sacituzumab Govitecan (IMMU-132). In addition, the anti-hTROP2 antibody-drug conjugates described in WO 2003/074566, WO 2011/068845, WO 2013/068946, or U.S. Patent No. 7,999,083 can also be used in the present invention.

Biological Samples Biological samples taken from a subject, e.g., a subject diagnosed with cancer, can be used as a source of RNA and the level of gene expression at the RNA level in the sample can be determined. The biological sample can include, for example, blood, e.g., whole blood, or blood derivatives, e.g., exosomes, tissues, cells, and/or circulatory tissue cells. In some embodiments, the biological sample can be removed from a tumor.

Exosomes are vesicles made of a lipid bilayer membrane secreted from cells. Since their discovery in the 1980s, many studies have revealed that they move between cells and transport a variety of molecules. Due to their morphological characteristics, they contain many physiologically active molecules, including not only proteins but also nucleic acids, carbohydrates, and lipids. It has also been revealed that exosomes contain miRNA and mRNA, which are transported between cells. Therefore, it is possible to select exosomes as a biological sample to which the present invention is applied.

Biological samples may be obtained by known means, such as venipuncture, or using known tumor biopsy devices and procedures. Endoscopic biopsy, excision biopsy, incision biopsy, fine needle biopsy, punch biopsy, excision biopsy, and skin biopsy are examples of recognized medical procedures that one of skill in the art may use to obtain a tumor sample. The biological sample should be large enough to provide sufficient RNA, or a thin section, for measuring gene expression.

In some embodiments, the methods include assessing gene expression at the mRNA level in a human subject diagnosed with cancer who provides an autologous tissue sample or consents to having an autologous tissue sample collected.

The biological sample may be in any form that allows for the measurement of gene expression or quantity. That is, the sample must be sufficient for RNA extraction or thin layer preparation. Thus, the sample may be fresh, preserved using appropriate cryogenic techniques, or preserved using non-cryotechniques. For example, a standard process for working with clinical biopsy samples is to fix the tissue sample in formalin and embed it in paraffin. Samples in this form are commonly known as formalin-fixed paraffin-embedded (FFPE) tissue. Suitable techniques for tissue preparation for subsequent analysis are well known to those skilled in the art.

Gene Expression In the present application, the determination or measurement of the gene expression level in a biological sample is carried out using a suitable method. Several such methods are well known in the art. For example, the determination of gene expression is made by measuring the level or amount of RNA, such as mRNA, in the sample.

PCR or microarray primers and/or probes are designed on the 3' end of the mRNA, which is believed to provide high preservation (stability) during the experimental process of RNA isolation or cDNA synthesis. The probes can be designed based on the desired sequence to detect specific forms of transcript variants. Examples of suitable detection methods include, but are not limited to, the following:

RNA Analysis Well-known microarray analysis and quantitative polymerase chain reaction (PCR) are examples of methods for determining the level of gene expression at the mRNA level. In some embodiments, RNA is extracted from cells, tumors, or tissues using standard protocols. In other embodiments, RNA analysis is performed using techniques that do not require RNA isolation.

Methods for rapid and efficient extraction of eukaryotic mRNA (i.e., poly(a)RNA) from tissue samples are well established and known to those skilled in the art. See, e.g., Ausubel et al., 1997, Current Protocols of Molecular Biology, John Wiley & Sons. The tissue sample may be a fresh, frozen, or fixed and paraffin-embedded (FFPE) clinical research tumor specimen. In general, RNA isolated from fresh or frozen tissue samples tends to be less fragmented than RNA from FFPE samples. However, FFPE samples of tumor material are more readily available and FFPE samples are a suitable source of RNA for use in the methods of the invention. For a discussion of FFPE samples as a source of RNA for gene expression profiling by RT-PCR, see, e.g., Clark-Langone et al., 2007, BMC Genomics 8:279. See also, De Andreus et al., 1995, Biotechniques 18:42044; and Baker et al., U.S. Patent Application Publication No. 2005/0095634.

The use of commercially available kits with manufacturer's instructions for RNA extraction and preparation is common. Commercial suppliers of various RNA isolation products and complete kits include Qiagen (Valencia, CA), Invitrogen (Carlsbad, CA), Ambion (Austin, TX), and Exiqon (Woburn, MA).

Generally, RNA isolation begins with tissue/cell disruption. During tissue/cell disruption, it is desirable to minimize RNA degradation by RNases. One approach to limit RNase activity during the RNA isolation process is to ensure that a denaturant is placed in contact with the cellular contents as soon as the cells are disrupted. Another common practice is to include one or more proteases in the RNA isolation process. Optionally, fresh tissue samples are immersed in an RNA stabilizing solution at room temperature as soon as they are collected. The stabilizing solution quickly penetrates the cells and stabilizes the RNA for storage at 4° C. and subsequent isolation. One such stabilizing solution is commercially available as RNAlater® (Ambion, Austin, TX).

In some protocols, total RNA is isolated from disrupted tumor material by cesium chloride density gradient centrifugation. In general, mRNA constitutes about 1%-5% of total cellular RNA. Immobilized oligo(dT) (e.g., oligo(dT) cellulose) is commonly used to separate mRNA from ribosomal and transfer RNA. If stored after isolation, RNA must be stored under RNase-free conditions. Methods for stable storage of isolated RNA are known in the art. A variety of commercial products for stable storage of RNA are available.

Microarrays mRNA expression levels can be determined (e.g., measured) using conventional DNA microarray expression profiling techniques. A DNA microarray is a collection of specific DNA segments or probes immobilized on a solid surface or support (e.g., glass, plastic, or silicon), with each specific DNA segment occupying a known position in the array. Typically, hybridization with a sample of labeled RNA under stringent conditions allows detection and quantification of the RNA molecules corresponding to each probe in the array. After stringent washing to remove non-specifically bound sample material, the microarray is scanned by confocal laser microscopy or other suitable detection methods. Modern commercially available DNA microarrays (often known as DNA chips) typically contain tens of thousands of probes and can therefore simultaneously measure the expression of tens of thousands of genes. Such microarrays can be used in the practice of the present invention. Alternatively, custom chips containing as many probes as are needed to measure the expression of specific genes and the necessary controls or standards (e.g., for data normalization) can be used in the practice of the present method.

To facilitate data normalization, a two-color microarray reader can be used. In a two-color (two-channel) system, the sample is labeled with a first fluorophore that emits at a first wavelength, while the RNA or cDNA standards are labeled with a second fluorophore that emits at a different wavelength. For example, Cy3 (570 nm) and Cy5 (670 nm) are often used together in a two-color microarray system.

DNA microarray technology is well developed, commercially available, and widely used. Thus, in practicing the present methods, one of skill in the art can use microarray technology to measure the expression levels of genes encoding biomarker proteins without undue experimentation. DNA microarray chips, reagents (e.g., preparation of RNA or cDNA, materials required for labeling RNA or cDNA, hybridization solutions, and washing solutions), equipment (e.g., microarray readers), and protocols are well known in the art and are commercially available from a variety of commercial sources. Commercial vendors of microarray systems include Agilent Technologies (Santa Clara, CA) and Affymetrix (Santa Clara, CA), although other array systems may be used.

Quantitative PCR
Levels of mRNA can be measured using conventional quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) technology. Advantages of qRT-PCR include sensitivity, flexibility, quantitative accuracy, and the ability to discriminate between mRNAs with high sequence identity. Guidance on processing tissue samples for quantitative PCR is available from a variety of sources, including manufacturers and suppliers of equipment and reagents for qRT-PCR, such as Qiagen (Valencia, CA) and Ambion (Austin, TX). Instruments and systems for automated operation of qRT-PCR are commercially available and are routinely used in many laboratories. An example of a well-known commercial system is the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

Once isolated mRNA is in hand, the first step in measuring gene expression by RT-PCR is to reverse transcribe the mRNA template into cDNA. The cDNA is then exponentially amplified in a PCR reaction. Two commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription reaction is typically primed with specific primers, random hexamers, or oligo(dT) primers. Suitable primers are commercially available (e.g., GeneAmp® RNA PCR kit (Perkin Elmer, Waltham, Mass.)). The resulting cDNA product can be used as a template in a subsequent polymerase chain reaction.

The PCR step is carried out using a thermostable DNA-dependent DNA polymerase. The most commonly used polymerase in PCR systems is Thermus aquaticus (Taq) polymerase. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification (i.e., the region of the cDNA reverse transcribed from the gene encoding the desired protein). Thus, when qRT-PCR is used in the present invention, primers specific for each marker gene are based on the cDNA sequence of the gene. Commercial techniques (e.g., SYBR® green or TaqMan® (Applied Biosystems, Foster City, Calif.)) can be used according to the manufacturer's instructions. mRNA levels can be normalized for loading differences between samples by comparing the levels of housekeeping genes (e.g., β-actin or GAPDH). The level of mRNA expression can be expressed relative to any single control sample (e.g., mRNA derived from normal non-tumor tissue or cells), or relative to mRNA derived from a pool of tumor samples, or tumor cell lines, or from a set of commercially available control mRNAs.

Suitable primer sets for PCR analysis of gene expression levels can be designed and synthesized by one of skill in the art without undue experimentation.

Alternatively, PCR primer sets for carrying out the invention can be purchased from commercial sources (e.g., Applied Biosystems). PCR primers are preferably about 17-25 nucleotides in length. Primers can be designed to have a specific melting temperature (Tm) using conventional algorithms for Tm estimation. Software for primer design and Tm estimation is commercially available (e.g., Primer ExpressTM (Applied Biosystems)) and also available on the internet (e.g., Primer3 (Massachusetts Institute of Technology)). By applying established principles of PCR primer design, a large number of different primers can be used to measure the expression level of any given gene.

qNPA
In some embodiments, RNA analysis is performed using techniques that do not involve RNA extraction or isolation. One such technique is the quantitative nuclease protection assay, commercially available under the name qNPA® (High Throughput Genomics, Inc., Tucson, AZ). This technique can be advantageous when the tissue sample being analyzed is in the form of FFPE material. See, e.g., See, e.g., Roberts et al, 2007, Laboratory Investigation 87:979-997.

nCounter Analysis System
nCounter (registered trademark) is a system for directly counting molecules based on digital molecular barcode technology developed by NanoString Technologies, and is capable of analyzing up to 800 types of RNA and DNA in a single tube quickly and with high accuracy. In the analysis by nCounter, a probe (Reporter Probe) having a barcode specific to the sequence of the target molecule and a probe (Capture Probe) for fixing to an analysis cartridge are hybridized with the target nucleic acid, and the sequence of the color barcode of each target sequence fixed to the surface of the cartridge is counted by a fluorescent scanner. For example, see Geiss G, et al., 26: 317-25 (2008)., Nature Biotechnology.

HTG EdgeSeq Assays
EdgeSeq is a sample profiling application, including tumor profiling, molecular diagnostic testing and biomarker development, consisting of instrumentation, consumables and software analysis developed by HTG Molecular Diagnostics. It automates molecular profiling of genes and gene activity using nuclease protection chemistry on biological samples. See, for example, Martel R., et al. Assay Drug Dev Technol. 2002 Nov;1(1):61-71. The expression levels of individual genes are obtained as count values by the above. The count values are used for analysis after a process called normalization, which corrects for distribution variations between samples. A specific normalization method can be mentioned as the median normalization method. In the median normalization method, a scaling factor is calculated for each sample by the method shown below, and the gene expression level is corrected by dividing the scaling factor. For the gth gene (Gene _g ) of the i-th sample (Sample _i ), the geometric mean of the expression levels (count values) of all samples is calculated, and the value obtained by dividing the expression level of Gene _g by the geometric mean is defined as the scaling factor ( _Sig ) for Gene _g of Sample _i . After the scaling factors are calculated for all genes, the median value of _Sig within Sample _i is defined as the scaling factor ( _Si ) of Sample _i . Finally, the expression levels of all genes in Sample _i are divided by S _i to obtain the Median Normalized Count (MNC). For details of this method, see, for example, Andres, S. and Huber W Genome Biol. 2010; 11(10):R106.

Next-generation RNA sequencing Unlike the conventional Sanger method, next-generation sequencing technology can obtain huge amounts of sequence information in a short time and at low cost by performing advanced parallel processing, and the expression of the entire transcriptome can be analyzed with higher sensitivity and accuracy. Representative next-generation sequencing technologies currently in widespread use include Illumina Inc.'s single-base synthesis reaction sequencing technology (Sequencing by synthesis) and Thermo Fisher Scientific Inc.'s ion semiconductor sequencing technology (Ion Torrent technology). For details of each technology, see, for example, Buermans HP., et al. Biochim Biophys Acta. 2014 Oct; 1842 (10): 1932-1941. In addition, each sequence information (read) obtained using the above-mentioned next-generation sequencing technology is used for analysis after undergoing a mapping operation to identify which gene transcript each read originates from and a normalization operation to correct the number of reads mapped to each transcript based on the length of the transcript and the total number of reads obtained in the analysis. A specific normalization method is, for example, the reads per kilobase of exon per million mapped sequence reads (RPKM) value, which is the number of reads corrected by the gene length of each gene, assuming that the length of the transcript is 1 kb and the total number of reads is 1 million. Similarly, the fragments per kilobase of exon per million mapped sequence reads (FPKM) value, which is the number of fragments corrected by the gene length of each gene with the total number of reads set to 1 million, and the transcripts per million (TPM) value, which represents the number of transcripts when the number of reads of each transcript is corrected by the gene length and the total number of reads is set to 1 million, are also widely used. RPKM uses a known gene model to count up the reads mapped to exons to calculate the expression level of each gene, while FPKM calculates the expression level of the isoform level by counting up the fragments for each estimated isoform. For details of each normalization method, see, for example, Conesa A., et al. Genome Biol. 2016 Jan 26;17:13.

Assessing hTROP2 Gene Expression hTROP2 gene expression may be assessed in a biological sample from a human patient. Such embodiments include requesting an assessment of hTROP2 gene expression at the mRNA level and receiving the assessment results. Some embodiments include determining the value of hTROP2 gene expression at the mRNA level and recording the value determined by any method.

The hTROP2 gene expression level may be interpreted in relation to a predetermined value. If the hTROP2 gene expression level is equal to, equal to, or exceeds the predetermined value, it is interpreted as predicting that the subject will be sensitive (responsive) to treatment with a pharmaceutical containing an anti-hTROP2 antibody. In some embodiments, if the hTROP2 gene expression level is equal to, equal to, or falls below the predetermined value, it is interpreted as predicting that the tumor will be resistant (non-responsive) to treatment with a pharmaceutical containing an anti-hTROP2 antibody.

In some embodiments, hTROP2 gene expression may be assessed as high or low based on a numerical value representing the level of hTROP2 gene expression in a biological sample. A subject may be assessed as high or low based on hTROP2 expression at the mRNA level, for example.

The expression level can be evaluated by any known method as described above. For example, the expression level of the hTROP2 gene can be evaluated based on the reads per kilobase of exon per million mapped sequence reads (RPKM) value calculated by next-generation RNA sequencing. The RPKM value is a value obtained by normalizing the number of reads obtained by a next-generation sequencer using the exon length of each gene and the total number of sequences read by the sequencer, and the expression level of the hTROP2 gene can be analyzed using the Log ₂ [RPKM+1] value, which is the logarithmic (Log ₂ ) value obtained by adding 1 to the RPKM value.

The RPKM value and hTROP2 gene expression are correlated. Thus, when the RPKM value is high, the hTROP2 gene expression is also high. In some embodiments, high hTROP2 gene expression is assessed when the _Log2 [RPKM+1] value is equal to or exceeds a predetermined value. The predetermined value can be statistically determined to minimize the undesirable effects of false positives and false negatives. The set numerical value can be selected in the range of 6.0 to 9.0, for example, from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0. The set numerical value can be selected in the range of 6.0 to 8.0, for example, from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The set numerical value can be selected in the range of 6.5 to 8.0, for example, from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. Furthermore, the set numerical value can be selected in the range of 7.0 to 8.0, for example, from the group consisting of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. Furthermore, the set numerical value can be selected in the range of 7.5 to 8.0, for example, from the group consisting of 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The set numerical value can also be selected from the group consisting of 6.5, 7.0, 7.5, and 8.0.

The expression level of the hTROP2 gene can be evaluated based on the fragments per kilobase of exon per million mapped sequence reads (FPKM) value calculated by next-generation RNA sequencing. The FPKM value is a value obtained by normalizing the number of reads obtained by a next-generation sequencer using the gene length of each gene and the total number of sequences read by the sequencer, and the expression level of the hTROP2 gene can be analyzed using the Log ₂ [FPKM+1] value, which is the logarithmic (Log ₂ ) value obtained by adding 1 to the FPKM value.

The FPKM value and hTROP2 gene expression are correlated. Thus, when the FPKM value is high, the hTROP2 gene expression is also high. In some embodiments, a high hTROP2 gene expression is evaluated when the Log ₂ [FPKM+1] value is equal to or exceeds a predetermined value. The predetermined value may be statistically set to minimize the undesirable effects of false positives and false negatives. The set value may be selected in the range of 6.0 to 8.0, for example, from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The set numerical value can be selected within the range of 6.0 to 7.0, for example, from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0. The set numerical value can also be selected as 6.0 or 7.0.

The expression level of the hTROP2 gene can be evaluated based on the Median Normalized Count (MNC) value calculated by EdgeSeq Assay. The MNC value is a value obtained by using the Median normalization method in EdgeSeq Assay, and the expression level of the hTROP2 gene can be analyzed using the Log ₂ [MNC+1] value, which is the logarithmic value (Log ₂ ) obtained by adding 1 to the MNC value.

The MNC value and the hTROP2 gene expression are correlated. Thus, when the MNC value is high, the hTROP2 gene expression is also high. In some embodiments, a high hTROP2 gene expression is evaluated when the Log ₂ [MNC+1] value is equal to or exceeds a predetermined value. The predetermined value may be statistically set to minimize the undesirable effects of false positives and false negatives. The set value may be selected in the range of 12.0 to 15.0, for example, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0. The value can be selected from the group consisting of 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, and 15.0. The set value can be selected from the range of 12.0 to 14.0, for example, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0. The set value can be selected from the group consisting of 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, and 14.0. The set value can also be selected as 12.0, 13.0, or 14.0.

In some embodiments, hTROP2 gene expression at the mRNA level is assessed using a regulatory agency approved test. In some embodiments, the regulatory agency approved test is a test approved by the FDA, EMA, or PMDA.

Assessing SLFN11 Gene Expression SLFN11 gene expression can be assessed in a biological sample from a human patient. Such embodiments include requesting an assessment of SLFN11 gene expression at the mRNA level and receiving the assessment results. Some embodiments include determining the value of SLFN11 gene expression at the mRNA level and recording the value determined by any method.

The SLFN11 gene expression level may be interpreted in relation to a predetermined value. If the SLFN11 gene expression level is equal to, equal to, or exceeds the predetermined value, it is interpreted as predicting that the subject will be sensitive (responsive) to treatment with a pharmaceutical containing an anti-hTROP2 antibody. In some embodiments, if the SLFN11 gene expression level is equal to, equal to, or falls below the predetermined value, it is interpreted as predicting that the tumor will be resistant (non-responsive) to treatment with a pharmaceutical containing an anti-hTROP2 antibody.

In some embodiments, SLFN11 gene expression can be assessed as high or low expression based on a numerical value representing the level of SLFN11 gene expression in a biological sample. A subject can be assessed as high or low expression based on SLFN11 expression at the mRNA level, for example.

The expression level can be evaluated by any known method as described above. For example, the expression level of SLFN11 gene can be evaluated based on the RPKM value, as in the case of hTROP2 gene. The expression level of SLFN11 gene can be analyzed using the Log ₂ [RPKM+1] value, which is the logarithmic value (Log ₂ ) obtained by adding 1 to the RPKM value.

The RPKM value and SLFN11 gene expression are correlated. Thus, when the RPKM value is high, the SLFN11 gene expression is also high. In some embodiments, a high SLFN11 gene expression is evaluated when the _Log2 [RPKM+1] value is above a predetermined value. The predetermined value can be statistically set to minimize the undesirable effects of false positives and false negatives. The set numerical value can be selected in the range of 1.0 to 4.0, for example, from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0. The set numerical value can be selected from the range of 1.0 to 3.0, for example, from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The set numerical value can be selected from the range of 2.0 to 3.0, for example, from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The set numerical value can be selected from the group consisting of 1.0, 2.0, and 3.0, and it is also possible to set it to 3.0.

The expression level of the SLFN11 gene can be evaluated based on the FPKM value, as in the case of the hTROP2 gene. The expression level of the SLFN11 gene can be analyzed using the Log ₂ [FPKM+1] value, which is the logarithmic value (Log ₂ ) obtained by adding 1 to the FPKM value.

The FPKM value and SLFN11 gene expression are correlated. Thus, when the FPKM value is high, the SLFN11 gene expression is also high. In some embodiments, a Log ₂ [FPKM] value above a predetermined value is evaluated as high SLFN11 gene expression. The predetermined value can be statistically set to minimize the undesirable effects of false positives and false negatives. The set value can be selected in the range of 2.0 to 4.0, for example, from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0. The set numerical value can be selected within the range of 2.0 to 3.0, and can be selected from the group consisting of, for example, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The set numerical value can be selected as 2.0 or 3.0.

The expression level of the SLFN11 gene can be evaluated based on the MNC value, as in the case of the hTROP2 gene. The expression level of the SLFN11 gene can be analyzed using the Log ₂ [MNC+1] value, which is the logarithmic value (Log ₂ ) obtained by adding 1 to the MNC value.

The MNC value and the SLFN11 gene expression are correlated. Thus, when the MNC value is high, the SLFN11 gene expression is also high. In some embodiments, a Log ₂ [MNC] value above a predetermined value is evaluated as high SLFN11 gene expression. The predetermined value can be statistically set to minimize the undesirable effects of false positives and false negatives. The set value can be selected in the range of 11.5 to 13.5, for example, from the group consisting of 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, and 13.5. The set numerical value can be selected within the range of 11.5 to 12.5, and can be selected from the group consisting of, for example, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, and 12.5. The set numerical value can be selected as 11.5, 12.0, or 12.5.

In some embodiments, SLFN11 gene expression at the mRNA level is assessed using a regulatory agency approved test. In some embodiments, the regulatory agency approved test is, for example, a test approved by the FDA, EMA, or PMDA.

Administration and Treatment In some embodiments, the evaluation of hTROP2 gene expression may be evaluated in combination with the expression of SLFN11 gene. By combining two types of sensitivity markers, more accurate evaluation is possible. In some embodiments, when the expression of hTROP2 gene and/or SLFN11 gene is evaluated to be high, a human patient suffering from cancer may be treated by administering a medicament containing an anti-hTROP2 antibody. In some embodiments, when the expression of hTROP2 gene and/or SLFN11 gene is evaluated to be low, a human patient suffering from cancer may be avoided from administering a medicament containing an anti-hTROP2 antibody.

Suitable doses of a pharmaceutical containing an anti-hTROP2 antibody include, but are not limited to, 2.0 mg/kg, 4.0 mg/kg, 6.0 mg/kg, 8.0 mg/kg, or 10.0 mg/kg. Suitable administration intervals of a pharmaceutical containing an anti-hTROP2 antibody include, but are not limited to, every 3 weeks.

The expression levels of hTROP2 and SLFN11 genes can be evaluated based on the RPKM value. The expression level of each gene can be analyzed using the _Log2 [RPKM+1] value, which is the logarithmic value ( _Log2 ) obtained by adding 1 to the RPKM value. The predetermined value can be statistically set to minimize the undesirable effects of false positives and false negatives.

As described above, the Log ₂ [RPKM+1] value in the hTROP2 gene can be set in the range of 6.0 to 9.0. Also, the Log ₂ [RPKM+1] value in the SLFN11 gene can be set in the range of 1.0 to 4.0. When the set values are combined, a human patient suffering from cancer can be treated by administering a medicament containing an anti-hTROP2 antibody when both the set values in the hTROP2 and SLFN11 genes are satisfied. Also, in some embodiments, a human patient can be treated by administering a medicament containing an anti-hTROP2 antibody when either one of the set values in the hTROP2 or SLFN11 genes is satisfied.

A combination of _Log2 [RPKM+1] set values suitable for the hTROP2 and SLFN11 genes includes a combination of a number selected from the group consisting of 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0 for the hTROP2 gene and a combination of a number selected from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 for the SLFN11 gene, and a combination of a number selected from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 for the hTROP2 gene and a combination of a number selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.7, 3.8, 3.9, 3.1, 3 Examples of the combination of the nucleotides include a combination of the nucleotides 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0, and a combination of the nucleotides 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 for the hTROP2 gene, and a combination of the nucleotides 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 for the SLFN11 gene.

Further combinations of suitable _Log2 [RPKM+1] set values in the hTROP2 and SLFN11 genes include a combination of a number selected from the group consisting of 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0 in the hTROP2 gene and a number selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 in the SLFN11 gene, a combination of a number selected from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 in the hTROP2 gene and a combination of a number selected from the group consisting of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 in the hTROP2 gene and a combination of a number selected from the group consisting of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, Examples of the combination of the nucleotide sequence of the present invention include a combination of the nucleotide sequence of the present invention selected from the group consisting of 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5 in the hTROP2 gene and a combination of the nucleotide sequence of the present invention selected from the group consisting of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0 in the SLFN11 gene, and a combination of the nucleotide sequence of the present invention selected from the group consisting of 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0 in the hTROP2 gene and a combination of the nucleotide sequence of the present invention selected from the group consisting of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0 in the SLFN11 gene.

More preferable combinations of Log ₂ [RPKM+1] set values in the hTROP2 and SLFN11 genes include a combination of 7.5 in the hTROP2 gene and 1.0 in the SLFN11 gene, or a combination of 6.5 in the hTROP2 gene and 2.0 in the SLFN11 gene.

Another more suitable combination of Log ₂ [RPKM+1] set values in the hTROP2 and SLFN11 genes can be a combination of 7.5 in the hTROP2 gene and 2.0 in the SLFN11 gene, or a combination of 6.5 in the hTROP2 gene and 3.0 in the SLFN11 gene.

Further preferred combinations of Log ₂ [RPKM+1] setting values in the hTROP2 and SLFN11 genes include any one combination selected from the group consisting of a combination of 7.5 in the hTROP2 gene and 1.0 in the SLFN11 gene, a combination of 6.5 in the hTROP2 gene and 2.0 in the SLFN11 gene, a combination of 7.5 in the hTROP2 gene and 2.0 in the SLFN11 gene, and a combination of 6.5 in the hTROP2 gene and 3.0 in the SLFN11 gene.

In addition, the expression levels of hTROP2 and SLFN11 genes can be evaluated based on the FPKM value. The expression level of each gene can be analyzed using a _Log2 [FPKM+1] value, which is the logarithmic value ( _Log2 ) obtained by adding 1 to the FPKM value. The predetermined value can be statistically set to minimize the undesirable effects of false positives and false negatives.

As described above, the Log ₂ [FPKM+1] value in the hTROP2 gene can be set in the range of 6.0 to 8.0. Also, the Log ₂ [FPKM+1] value in the SLFN11 gene can be set in the range of 2.0 to 4.0. When the set values are combined, a human patient suffering from cancer can be treated by administering a medicament containing an anti-hTROP2 antibody when both the set values in the hTROP2 and SLFN11 genes are satisfied. Also, in some embodiments, a human patient can be treated by administering a medicament containing an anti-hTROP2 antibody when either one of the set values in the hTROP2 or SLFN11 genes is satisfied.

A suitable combination of Log ₂ [FPKM+1] set values for the hTROP2 and SLFN11 genes is as follows:
For the hTROP2 gene, examples include a combination of one number selected from the group consisting of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and for the SLFN11 gene, examples include a combination of one number selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.

Another preferred combination is a combination of a number selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 for the hTROP2 gene, and a combination of a number selected from the group consisting of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0 for the SLFN11 gene.

Yet another example of a combination is one number selected from the group consisting of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0 for the hTROP2 gene, and one number selected from the group consisting of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0 for the SLFN11 gene.

Other combinations of more preferred Log ₂ [FPKM+1] setting values in the hTROP2 and SLFN11 genes include any one combination selected from the group consisting of a combination of 7.0 in the hTROP2 gene and 2.0 in the SLFN11 gene, a combination of 6.0 in the hTROP2 gene and 3.0 in the SLFN11 gene, and a combination of 7.0 in the hTROP2 gene and 3.0 in the SLFN11 gene.

Furthermore, the expression levels of hTROP2 and SLFN11 genes can be evaluated based on the MNC value. The expression level of each gene can be analyzed using the Log ₂ [MNC+1] value, which is the logarithm (Log ₂ ) of the MNC value plus 1. The predetermined value can be statistically set to minimize the undesirable effects of false positives and false negatives.

As described above, the Log ₂ [MNC+1] value in the hTROP2 gene can be set in the range of 12.0 to 15.0 . Also, the Log ₂ [MNC+1] value in the SLFN11 gene can be set in the range of 11.5 to 13.5 . When the set values are combined, a human patient suffering from cancer can be treated by administering a medicament containing an anti-hTROP2 antibody when both the set values in the hTROP2 and SLFN11 genes are satisfied. Also, in some embodiments, a human patient can be treated by administering a medicament containing an anti-hTROP2 antibody when either one of the set values in the hTROP2 or SLFN11 genes is satisfied.

Suitable combinations of Log ₂ [MNC+1] set values for the hTROP2 and SLFN11 genes are 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, and 13.0 for the hTROP2 gene. and for the SLFN11 gene, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, An example of the combination of the numbers is one selected from the group consisting of 13.4, 13.5, and 13.6.

Another preferred combination is a combination of a number selected from the group consisting of 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, and 14.0 for the hTROP2 gene, and a combination of a number selected from the group consisting of 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, and 12.5 for the SLFN11 gene.

Yet another example of a combination is a combination of a number selected from 12.0, 13.0, or 14.0 for the hTROP2 gene and a number selected from 11.5, 12.0, or 12.5 for the SLFN11 gene.

Further preferred combinations of Log ₂ [MNC+1] set values in the hTROP2 and SLFN11 genes include any one combination selected from the group consisting of a combination of 12.0 in the hTROP2 gene and 11.5 in the SLFN11 gene, a combination of 14.0 in the hTROP2 gene and 11.5 in the SLFN11 gene, a combination of 12.0 in the hTROP2 gene and 12.5 in the SLFN11 gene, and a combination of 14.0 in the hTROP2 gene and 12.5 in the SLFN11 gene.

Measurement and evaluation of gene expression levels may be performed simultaneously for the hTROP2 gene and the SLFN11 gene.

Alternatively, the expression level of the hTROP2 gene may be measured and evaluated first, and then the expression level of the SLFN11 gene may be measured and evaluated in human patients who are evaluated to have a high expression level of the hTROP2 gene.

Alternatively, the expression level of the SLFN11 gene may be measured and evaluated first, and then the expression level of the hTROP2 gene may be measured and evaluated in human patients who are evaluated to have a high expression level of the SLFN11 gene.

In some embodiments, hTROP2 and SLFN11 gene expression at the mRNA level is assessed using a regulatory agency approved test. In some embodiments, the regulatory agency approved test is an FDA, EMA, or PMDA approved test.

Test Kits The present invention also relates to diagnostic test kits comprising several components for carrying out the methods of the present invention. Diagnostic test kits increase the convenience, speed and reproducibility of performing diagnostic assays. For example, in qRT-PCR based embodiments, a basic diagnostic test kit includes PCR primers for analyzing gene expression. In other embodiments, more detailed test kits include PCR primers as well as buffers, reagents and detailed instructions for measuring gene expression levels using PCR technology. In some embodiments, the kits include all consumable components required for testing, other than the test protocol and the RNA sample.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.
Example 1. Production of antibody-drug conjugates According to the production methods described in WO 2015/098099 and WO 2017/002776, a humanized anti-hTROP2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 2) was used to produce an antibody-drug conjugate of the formula:

(In the formula, A represents the binding site to the anti-hTROP2 antibody.)
An antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (1)") was produced in which a drug linker represented by the formula: is linked to an anti-hTROP2 antibody via a thioether bond. The average number of drugs bound per antibody molecule can be adjusted within the range of 0 to 8, but in this example, an antibody-drug conjugate with an average number of drugs bound of 3.5 to 4.5 was produced and used in the following examples.

Example 2. Evaluation of antitumor effect of antibody-drug conjugates (1)
Mice: 5-8 week-old female nu/nu mice (Envigo) were acclimated to SPF conditions for at least 3 days before use in the experiment. Mice were fed sterilized solid food (Teklad 2919, Envigo) and provided with reverse osmosis treated water (containing 2 ppm chlorine).

Measurement and calculation formula: The major and minor axes of the tumor were measured twice a week using an electronic digital caliper (CD-6PMX, Mitutoyo Corp.), and the tumor volume (mm ³ ) was calculated using the following formula.
Tumor volume (mm ³ ) = 0.52 × major axis (mm) × [minor axis (mm)] ²
Tumor pieces derived from each cancer patient were subcutaneously transplanted and passaged into mice, and the tumor pieces were cut into ^pieces of approximately 5x5x5 mm3 and transplanted subcutaneously into the left flank of female nu/nu mice to create patient-derived xenograft (PDX) models. When the average volume of the transplanted tumors reached 150-300 ^mm3 , the mice were randomly divided into groups (Day 0). On the same day as grouping, antibody-drug conjugate (1) was administered into the tail vein at a dose of 10 mg/kg. As a negative control, formulation buffer was administered in the same manner at a liquid volume of 10 mL/kg. Using the tumor volumes of each tumor 21 days after administration, the tumor growth inhibition rate (TGI (%)) was calculated according to the following formula (Table 1).
Tumor growth inhibition rate (%) = 100 × (1-Tf/mean Cf)
Tf: mean tumor volume 21 days after administration of antibody-drug conjugate (1) Cf: arithmetic mean tumor volume 21 days after administration of negative control mice All of the above tests were performed at Champions Oncology (Champions).

Example 3. Relationship between hTROP2 and SLFN11 gene expression levels (RPKM value) in tumors derived from each PDX mouse model and the antitumor activity of antibody-drug conjugate (1) The gene expression data in each PDX model used in Example 2 was obtained by adding 1 to the RPKM value obtained and normalized by Champions, and taking the logarithm (Log ₂ ), i.e., Log ₂ [RPKM+1] value (Table 2), and the relationship between the antitumor activity (Table 1) of antibody-drug conjugate (1) in the PDX model and the expression levels of the hTROP2 gene and the SLFN11 gene was analyzed using this. When all the models evaluated were divided into groups showing a certain level or more of expression of the hTROP2 gene and the SLFN11 gene (Table 3), it was shown that the proportion of animal models showing a certain level or more of efficacy (in this case, the standard of TGI of 75% or more was adopted as an example) increased with increasing hTROP2 gene expression and increasing SLFN11 gene expression (Table 4). When grouping based on SLFN11 gene expression was not performed, the proportion of animal models showing a TGI of 75% or more was only 47-63%, and it was revealed that a combination of the hTROP2 gene expression level and the SLFN11 gene expression level can be used as a sensitive marker for predicting the antitumor effect of antibody-drug conjugate (1). For example, when the Log ₂ [RPKM+1] value of the hTROP2 gene is greater than 7.5 and the Log ₂ [RPKM+1] value of the SLFN11 gene is greater than 1.0, or when the Log ₂ [RPKM+1] value of the hTROP2 gene is greater than 6.5 and the Log ₂ [RPKM+1] value of the SLFN11 gene is greater than 2.0, the proportion of animal models showing a TGI of 75% or more is about 80% to 100%. In addition, when the Log ₂ [RPKM+1] value of the hTROP2 gene exceeds 7.5 and the Log ₂ [RPKM+1] value of the SLFN11 gene exceeds 2.0, or when the Log ₂ [RPKM+1] value of the hTROP2 gene exceeds 6.5 and the Log ₂ [RPKM+1] value of the SLFN11 gene exceeds 3.0, the proportion of animal models showing a TGI of 75% or more is 100%. Even when the TGI is 60% or more or 70% or more, it was possible to predict the antitumor effect with the same prediction rate by each Log ₂ [RPKM+1] value set when the TGI is 75% or more. When TGI was 80% or more, the prediction rate dropped to the 60% range only when the Log ₂ [RPKM+1] value was greater than 7.5 and the Log ₂ [RPKM+1] value of the SLFN11 gene was greater than 1.0 and less than or equal to 2.

Example 4. Evaluation of antitumor effect of antibody-drug conjugates (2)
Mice: 5-8 week-old female nu/nu mice (Envigo) were acclimated to SPF conditions for at least 3 days before use in the experiment. Mice were fed sterilized solid food (Teklad 2919, Envigo) and provided with reverse osmosis treated water (containing 2 ppm chlorine).

Measurement and calculation formula: The major and minor axes of the tumor were measured twice a week using an electronic digital caliper (CD-6PMX, Mitutoyo Corp.), and the tumor volume (mm ³ ) was calculated using the following formula.
Tumor volume (mm ³ ) = 0.52 × major axis (mm) × [minor axis (mm)] ²
Tumor pieces derived from each cancer patient were subcutaneously transplanted and passaged into mice, and the tumor pieces were cut into ^pieces of approximately 5x5x5 mm3 and transplanted subcutaneously into the left flank of female nu/nu mice to create patient-derived xenograft (PDX) models. When the average volume of the transplanted tumor reached 150-300 ^mm3 , the mice were randomly divided into groups (Day 0). On the same day as grouping, antibody-drug conjugate (1) was administered into the tail vein at a dose of 10 mg/kg. As a negative control, formulation buffer was administered in a similar manner at a liquid volume of 10 mL/kg.
For the PDX models evaluated in Example 2 and this Example, the tumor growth inhibition rate (TGI (%)) was calculated according to the following formula using the tumor volumes 10 to 15 days after administration (Table 5).
Tumor growth inhibition rate (%) = 100 × (1-Tf/mean Cf)
Tf: mean tumor volume 10-15 days after administration of antibody-drug conjugate (1) Cf: arithmetic mean tumor volume 10-15 days after administration of negative control mice

Example 5. Relationship between hTROP2 and SLFN11 gene expression levels (FPKM value) in tumors derived from each PDX mouse model and the antitumor activity of antibody-drug conjugate (1) Gene expression data in each PDX model used in Example 4 was obtained by next-generation RNA sequencing using RNA extracted from formalin-fixed paraffin-embedded specimens of each tumor. The obtained data was normalized to the FPKM value, and then 1 was added to the FPKM to obtain a logarithmic (Log ₂ ) value, Log ₂ [FPKM+1] (Table 6), which was used to analyze the relationship between the antitumor activity of antibody-drug conjugate (1) in the PDX model (Table 5) and the expression levels of the hTROP2 gene and the SLFN11 gene. When all the evaluated models were divided into groups in which the hTROP2 gene and the SLFN11 gene showed a certain level of expression or more (Table 7), it was shown that the percentage of animal models showing a certain level of efficacy or more (TGI of 75% or more was used as an example in this case) increased with an increase in hTROP2 gene expression and an increase in SLFN11 gene expression (Table 8). When grouping based on SLFN11 gene expression was not performed, the percentage of animal models showing a TGI of 75% or more was only 43-50%, and it was revealed that the combination of the hTROP2 gene expression level and the SLFN11 gene expression level can be used as a sensitivity marker for predicting the antitumor effect of the antibody-drug conjugate (1). For example, when the _Log2 [FPKM+1] value of the hTROP2 gene is greater than 7.0 and the _Log2 [FPKM+1] value of the SLFN11 gene is greater than 2.0, or when the _Log2 [FPKM+1] value of the hTROP2 gene is greater than 6.0 and the _Log2 [FPKM+1] value of the SLFN11 gene is greater than 3.0, the percentage of animal models showing a TGI of 75% or more is about 80% to 100%. Also, when the _Log2 [FPKM+1] value of the hTROP2 gene is greater than 7.0 and the _Log2 [FPKM+1] value of the SLFN11 gene is greater than 3.0, the percentage of animal models showing a TGI of 75% or more is 100%. Even when the TGI was 70% or 80% or more, it was possible to predict the antitumor effect with the same prediction rate using each _Log2 [FPKM+1] value set when the TGI was 75% or more.

Example 6. Preparation of Compound (1) According to the preparation methods described in WO 2014/057687 and WO 2015/115091, a compound of the formula

We produced a compound represented by the formula (hereinafter referred to as "compound (1)").

Example 7. Cell proliferation inhibition test upon SLFN11 knockdown 7-(1): Effect on human pharyngeal cancer cell line FaDu Human pharyngeal cancer cell line FaDu was obtained from ATCC and used for evaluation. FaDu cells suspended at 2 x 10 ⁵ cells/mL in MEM medium containing non-essential amino acid solution, pyruvic acid solution, and 10% fetal bovine serum were seeded on a 10 cm cell culture dish at 10 mL/dish. 24 hours after seeding, 100 pmol of ON-TARGETplus SLFN11 siRNA (Dharamcon) or ON-TARGETplus Non-targeting Control Pool (Dharamcon) and 30 μL of Lipofectamine ^™ RNAiMAX Transfection Reagent (ThermoFisher) were suspended in 1 mL of Opti-MEM medium and the entire amount was added to the medium. After 72 hours, the medium was removed and washed with PBS, and the cells were dissociated from the dish using 1 mL of TrypLE ^™ Express and collected. The collected cells were suspended at 5 x 10 ⁴ cells/mL in MEM medium containing non-essential amino acid solution, pyruvic acid solution and 10% fetal bovine serum. They were seeded at 100 μL/well in a 96-well cell culture plate. 24 hours after seeding, the medium was replaced with medium containing 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.13 nM, 0.045 nM, or 0 nM of compound (1) or antibody-drug conjugate (1) at 100 μL/well. The molar concentration of antibody-drug conjugate (1) was calculated assuming an average molecular weight of 150,000. All cells were cultured at 37°C under 5% CO ₂ . 72 hours after the medium change, ATPlite 1step detection system (PerkinElmer) was added at 100 μL/well, and after incubation at room temperature for 10 minutes, the luminescence intensity of each well was measured.

The cell proliferation inhibition rate (%) under each condition was calculated using the following formula.
Cell proliferation inhibition rate (%) = 100 x (1 - T/C)
T: Average luminescence intensity of each well to which a sample was added C: Average luminescence intensity of each well to which 0 nM of a sample was added

The 50% inhibitory concentration under each condition was calculated by fitting to the following formula.
Cell viability (%) = (Emax - Emin) x sample concentration ^γ /
((Emax-50)/(50-Emin) x IC50 ^γ + sample concentration ^γ )
+ Emin
Emax: Maximum cell proliferation inhibition rate (%)
Emin: Minimum cell proliferation inhibition rate (%)
IC50: 50% inhibitory concentration γ: Fitting to the Hill coefficient calculation formula was performed using SAS system Release 9.2 (SAS Institute Inc.).

The cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in FaDu cells upon SLFN11 knockdown is shown in Figure 4. The calculated 50% inhibitory concentrations are shown in Table 9. The cell proliferation inhibitory effect of compound (1) and antibody-drug conjugate (1) on the FaDu cell line was attenuated by SLFN11 knockdown.

7-(2): Effect on human lung cancer cell line NCI-H1781 Human lung cancer cell line NCI-H1781 was obtained from ATCC and used for evaluation. NCI-H1781 cells suspended at 2 x 10 ⁵ cells/mL in RPMI-1640 medium containing 10% fetal bovine serum were seeded on 10 cm cell culture dishes at 10 mL/dish. 24 hours after seeding, 100 pmol of ON-TARGETplus SLFN11 siRNA (Dharamcon) or ON-TARGETplus Non-targeting Control Pool (Dharamcon) and 30 μL of Lipofectamine ^™ RNAiMAX Transfection Reagent (ThermoFisher) were suspended in 1 mL of Opti-MEM medium and the entire amount was added to the medium. After 72 hours, the medium was removed and washed with PBS, and the cells were dissociated from the dish using 1 mL of TrypLE ^™ Express and collected. The collected cells were suspended at 5 x 10 ⁴ cells/mL in RPMI-1640 medium containing 10% fetal bovine serum. They were seeded at 100 μL/well in a 96-well cell culture plate. 24 hours after seeding, the medium was replaced with medium containing 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.13 nM, 0.045 nM, or 0 nM of compound (1) or antibody-drug conjugate (1) at 100 μL/well. The molar concentration of antibody-drug conjugate (1) was calculated assuming an average molecular weight of 150,000. All cells were cultured at 37°C under 5% CO ₂ . 72 hours after the medium change, ATPlite 1step detection system (PerkinElmer) was added at 100 μL/well, and after incubation at room temperature for 10 minutes, the luminescence intensity of each well was measured.

The cell proliferation inhibition rate (%) under each condition was calculated using the following formula.
Cell proliferation inhibition rate (%) = 100 x (1 - T/C)
T: Average luminescence intensity of each well to which a sample was added C: Average luminescence intensity of each well to which 0 nM of a sample was added

The 50% inhibitory concentration under each condition was calculated by fitting to the following formula.
Cell viability (%) = (Emax - Emin) x sample concentration ^γ /
((Emax-50)/(50-Emin) x IC50 ^γ + sample concentration ^γ )
+ Emin
Emax: Maximum cell proliferation inhibition rate (%)
Emin: Minimum cell proliferation inhibition rate (%)
IC50: 50% inhibitory concentration γ: Hill coefficient

SAS system Release 9.2 (SAS Institute Inc.) was used for fitting to the formula.

The cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in NCI-H1781 cells when SLFN11 was knocked down is shown in Figure 5. The calculated 50% inhibitory concentrations are shown in Table 10. The cell proliferation inhibitory effect of compound (1) and antibody-drug conjugate (1) in the NCI-H1781 cell line was attenuated by SLFN11 knockdown.

7-(3): Effect on human lung cancer cell line Calu-3 Human lung cancer cell line Calu-3 was obtained from ATCC and used for evaluation. Calu-3 cells suspended at 2 x ¹⁰⁵ cells/mL in MEM medium containing non-essential amino acid solution, pyruvic acid solution, and 10% fetal bovine serum were seeded on 10 cm cell culture dishes at 10 mL/dish. 24 hours after seeding, 100 pmol of ON-TARGETplus SLFN11 siRNA (Dharamcon) or ON-TARGETplus Non-targeting Control Pool (Dharamcon) and 30 μL of Lipofectamine ^™ RNAiMAX Transfection Reagent (ThermoFisher) were suspended in 1 mL of Opti-MEM medium and the entire amount was added to the medium. After 72 hours, the medium was removed and washed with PBS, and the cells were dissociated from the dish using 1 mL of TrypLE ^™ Express and collected. The collected cells were suspended at 5 x 10 ⁴ cells/mL in MEM medium containing non-essential amino acid solution, pyruvic acid solution and 10% fetal bovine serum. They were seeded at 100 μL/well in a 96-well cell culture plate. 24 hours after seeding, the medium was replaced with medium containing 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.13 nM, 0.045 nM, or 0 nM of compound (1) or antibody-drug conjugate (1) at 100 μL/well. The molar concentration of antibody-drug conjugate (1) was calculated assuming an average molecular weight of 150,000. All cells were cultured at 37°C under 5% CO ₂ . 72 hours after the medium change, ATPlite 1step detection system (PerkinElmer) was added at 100 μL/well, and after incubation at room temperature for 10 minutes, the luminescence intensity of each well was measured.

The cell proliferation inhibition rate (%) under each condition was calculated using the following formula.
Cell proliferation inhibition rate (%) = 100 x (1 - T/C)
T: Average luminescence intensity of each well to which a sample was added C: Average luminescence intensity of each well to which 0 nM of a sample was added

The 50% inhibitory concentration under each condition was calculated by fitting to the following formula.
Cell viability (%) = (Emax - Emin) x sample concentration ^γ /
((Emax-50)/(50-Emin) x IC50 ^γ + sample concentration ^γ )
+ Emin
Emax: Maximum cell proliferation inhibition rate (%)
Emin: Minimum cell proliferation inhibition rate (%)
IC50: 50% inhibitory concentration γ: Hill coefficient

SAS system Release 9.2 (SAS Institute Inc.) was used for fitting to the formula.

The cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in Calu-3 cells when SLFN11 was knocked down is shown in Figure 6. The calculated 50% inhibitory concentrations are shown in Table 11. The cell proliferation inhibitory effect of compound (1) in the Calu-3 cell line was attenuated by SLFN11 knockdown.

7-(4): Effect on human breast cancer cell line MDA-MB-468 Human breast cancer cell line MDA-MB-468 was obtained from ATCC and used for evaluation. MDA-MB-468 cells suspended at 2 x 10 ⁵ cells/mL in RPMI-1640 medium containing 10% fetal bovine serum were seeded on 10 cm cell culture dishes at 10 mL/dish. 24 hours after seeding, 100 pmol of ON-TARGETplus SLFN11 siRNA (Dharamcon) or ON-TARGETplus Non-targeting Control Pool (Dharamcon) and 30 μL of Lipofectamine ^™ RNAiMAX Transfection Reagent (ThermoFisher) were suspended in 1 mL of Opti-MEM medium and the entire amount was added to the medium. After 72 hours, the medium was removed and washed with PBS, and the cells were dissociated from the dish using 1 mL of TrypLE ^™ Express and collected. The collected cells were suspended at 5 x 10 ⁴ cells/mL in RPMI-1640 medium containing 10% fetal bovine serum. They were seeded at 100 μL/well in a 96-well cell culture plate. 24 hours after seeding, the medium was replaced with medium containing 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.13 nM, 0.045 nM, or 0 nM of compound (1) or antibody-drug conjugate (1) at 100 μL/well. The molar concentration of antibody-drug conjugate (1) was calculated assuming an average molecular weight of 150,000. All cells were cultured at 37°C under 5% CO ₂ . 72 hours after the medium change, ATPlite 1step detection system (PerkinElmer) was added at 100 μL/well, and after incubation at room temperature for 10 minutes, the luminescence intensity of each well was measured.

The cell proliferation inhibition rate (%) under each condition was calculated using the following formula.
Cell proliferation inhibition rate (%) = 100 x (1 - T/C)
T: Average luminescence intensity of each well to which a sample was added C: Average luminescence intensity of each well to which 0 nM of a sample was added

The 50% inhibitory concentration under each condition was calculated by fitting to the following formula.
Cell viability (%) = (Emax - Emin) x sample concentration ^γ /
((Emax-50)/(50-Emin) x IC50 ^γ + sample concentration ^γ )
+ Emin
Emax: Maximum cell proliferation inhibition rate (%)
Emin: Minimum cell proliferation inhibition rate (%)
IC50: 50% inhibitory concentration γ: Hill coefficient

SAS system Release 9.2 (SAS Institute Inc.) was used for fitting to the formula.

The cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) in MDA-MB-468 cells upon SLFN11 knockdown is shown in Figure 7. The calculated 50% inhibitory concentrations are shown in Table 12. The cell proliferation inhibitory effect of compound (1) on the MDA-MB-468 cell line was attenuated by SLFN11 knockdown.

7-(5): Effect on human breast cancer cell line HCC38 Human breast cancer cell line HCC38 was obtained from ATCC and used for evaluation. HCC38 cells suspended at 2 x 10 ⁵ cells/mL in RPMI-1640 medium containing 10% fetal bovine serum were seeded on 10 cm cell culture dishes at 10 mL/dish. 24 hours after seeding, 100 pmol of ON-TARGETplus SLFN11 siRNA (Dharamcon) or ON-TARGETplus Non-targeting Control Pool (Dharamcon) and 30 μL of Lipofectamine ^™ RNAiMAX Transfection Reagent (ThermoFisher) were suspended in 1 mL of Opti-MEM medium and the entire amount was added to the medium. After 72 hours, the medium was removed and washed with PBS, and the cells were dissociated from the dish using 1 mL of TrypLE ^™ Express and collected. The collected cells were suspended at 5 x 10 ⁴ cells/mL in RPMI-1640 medium containing 10% fetal bovine serum. They were seeded at 100 μL/well in a 96-well cell culture plate. 24 hours after seeding, the medium was replaced with medium containing 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.13 nM, 0.045 nM, or 0 nM of compound (1) or antibody-drug conjugate (1)a at 100 μL/well. The molar concentration of antibody-drug conjugate (1) was calculated assuming an average molecular weight of 150,000. All cells were cultured at 37°C under 5% CO ₂ . 72 hours after the medium change, ATPlite 1step detection system (PerkinElmer) was added at 100 μL/well, and after incubation at room temperature for 10 minutes, the luminescence intensity of each well was measured.

The cell proliferation inhibition rate (%) under each condition was calculated using the following formula.
Cell proliferation inhibition rate (%) = 100 x (1 - T/C)
T: Average luminescence intensity of each well to which a sample was added C: Average luminescence intensity of each well to which 0 nM of a sample was added

The 50% inhibitory concentration under each condition was calculated by fitting to the following formula.
Cell viability (%) = (Emax - Emin) x sample concentration ^γ /
((Emax-50)/(50-Emin) x IC50 ^γ + sample concentration ^γ )
+ Emin
Emax: Maximum cell proliferation inhibition rate (%)
Emin: Minimum cell proliferation inhibition rate (%)
IC50: 50% inhibitory concentration γ: Hill coefficient

SAS system Release 9.2 (SAS Institute Inc.) was used for fitting to the formula.

The cell proliferation inhibitory effect of compound (1) or antibody-drug conjugate (1) on HCC38 cells when SLFN11 was knocked down is shown in Figure 8. The calculated 50% inhibitory concentrations are shown in Table 13. The cell proliferation inhibitory effect of compound (1) and antibody-drug conjugate (1) on the HCC38 cell line was weakened by SLFN11 knockdown.

Example 8. hTROP2 and SLFN11 gene expression levels (Median Normalized Count values) of tumors derived from each patient in a clinical trial, and their relationship with the antitumor activity of antibody-drug conjugate (1) 8-(1) Study design and drug effects In the dose escalation part of a Phase 1 clinical trial targeting patients with recurrent or progressive non-small cell lung cancer, antibody-drug conjugate (1) was administered intravenously once every three weeks until unacceptable toxicity or progression of the disease was observed. Dose-limiting toxicity was determined in Cycle 1 (Day 1-21). Tumor sampling was performed before the first dose after entry into the clinical trial. The administered dose and maximum tumor change rate (%) for each patient are shown in Table 14. Note that a negative value for the maximum tumor change rate means that the tumor was reduced by administration of antibody-drug conjugate (1).

8-(2) Measurement of SLFN11 and TROP2 mRNA levels in tumors Gene expression data for each patient was obtained by cutting a section from a formalin-fixed paraffin-embedded specimen of tumor tissue of each patient before administration of the antibody-drug conjugate (1), excising the tumor site by laser microdissection, and then using EdgeSeq. The obtained count data was normalized by the Median Normalization method to obtain the expression level of each gene. All of the above tests were performed at HTG Molecular Diagnostics.

Log ₂ [MNC+1] values were obtained by adding 1 to the Median Normalized Count (MNC) values obtained and normalized by HTG Molecular Diagnostics to obtain logarithmic (Log ₂ ) values (Table 15), and the relationship between the antitumor activity of the antibody-drug conjugate (1) in patients (Table 14) and the expression levels of the hTROP2 gene and the SLFN11 gene was analyzed using the values. When all evaluated patients were divided into groups showing a certain level or higher of expression of the hTROP2 gene and the SLFN11 gene (Table 16), it was shown that the proportion of patients showing a certain level or higher of efficacy (maximum tumor change rate of 0% or less) increased with increasing hTROP2 gene expression and increasing SLFN11 gene expression (Table 17). When grouping based on SLFN11 gene expression was not performed, the proportion of patients showing a maximum tumor change rate of 0% or less was only 75-80%, and it was revealed that a combination of the hTROP2 gene expression level and the SLFN11 gene expression level can be used as a sensitive marker for predicting the antitumor effect of antibody-drug conjugate (1). For example, when the Log ₂ [MNC+1] value of the hTROP2 gene exceeds 12 and the Log ₂ [MNC+1] value of the SLFN11 gene exceeds 11.5, the proportion of patients showing a maximum tumor change rate of 0% or less is about 80% to 100%.

SEQ ID NO:1: Amino acid sequence of the humanized anti-hTROP2 antibody heavy chain SEQ ID NO:2: Amino acid sequence of the humanized anti-hTROP2 antibody light chain SEQ ID NO:3: CDRH1 sequence of the humanized anti-hTROP2 antibody heavy chain SEQ ID NO:4: CDRH2 sequence of the humanized anti-hTROP2 antibody heavy chain SEQ ID NO:5: CDRH3 sequence of the humanized anti-hTROP2 antibody heavy chain SEQ ID NO:6: CDRL1 sequence of the humanized anti-hTROP2 antibody light chain SEQ ID NO:7: CDRL2 sequence of the humanized anti-hTROP2 antibody light chain SEQ ID NO:8: CDRL3 sequence of the humanized anti-hTROP2 antibody light chain

Claims (52)

A method for identifying a subject to be administered a medicament containing an anti-hTROP2 antibody in a human patient suffering from cancer, comprising:
1 ) assessing the expression level of the hTROP2 gene at the mRNA level in a biological sample obtained from a human patient diagnosed with cancer ;
2 ) evaluating the expression level of the SLFN11 gene at the mRNA level in the biological sample in which the expression level of the hTROP2 gene has been determined to be high; and
3 ) identifying a human patient having the biological sample in which the expression level of the SLFN11 gene has been determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes: A method for identifying a subject to be administered a medicament containing an anti-hTROP2 antibody in a human patient suffering from cancer, comprising:
1 ) evaluating the expression levels of the hTROP2 gene and the SLFN11 gene at the mRNA level in a biological sample obtained from a human patient diagnosed with cancer ; and
2 ) identifying a human patient having a sample in which the expression levels of the hTROP2 gene and the SLFN11 gene have been determined to be high, as a subject to be administered a pharmaceutical agent containing an anti-hTROP2 antibody;
The method includes: The method according to claim 1 or 2, wherein the log2[RPKM+1] value is measured by RNA sequencing from a biological sample obtained from a human patient diagnosed with cancer, and if the log2[RPKM+1] value exceeds a specific value, the expression level of the hTROP2 gene and/or the SLFN11 gene at the mRNA level is determined to be high. The method according to claim 3, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0. 5. The method according to claim 3 or 4, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The method according to any one of claims 3 to 5, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The method according to any one of claims 3 to 6, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value exceeds any one selected from the group consisting of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. 8. The method according to any one of claims 3 to 7, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The method according to any one of claims 3 to 7, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 7.0, 7.5, and 8.0. The method according to claim 9, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than 7.0. The method according to claim 9, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than 7.5. The method according to claim 9, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than 8.0. The method according to any one of claims 3 to 12, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0. The method according to any one of claims 3 to 13, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The method according to any one of claims 3 to 14, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than any one selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The method according to any one of claims 3 to 14, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[RPKM+1] value exceeds any one selected from the group consisting of 1.0, 2.0, and 3.0. The method according to claim 16, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than 1.0. The method according to claim 16, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[RPKM+1] value is greater than 2.0. SLFN1 at the mRNA level when the log2[RPKM+1] value is greater than 3.0
The method according to claim 16, wherein the expression level of one gene is determined to be high. The method according to claim 1 or 2, wherein the log2[FPKM+1] value is measured by RNA sequencing from a biological sample obtained from a human patient diagnosed with cancer, and if the log2[FPKM+1] value exceeds a specific value, the expression level of the hTROP2 gene and/or the SLFN11 gene at the mRNA level is determined to be high. 21. The method according to claim 20, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. The method according to claim 20 or 21, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than any one selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0. The method according to any one of claims 20 to 22 , wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 6.0 or 7.0. The method according to claim 23 , wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 6.0. The method according to claim 23 , wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 7.0. The method according to any one of claims 20 to 25, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than any one selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0. The method according to any one of claims 20 to 26, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than any one selected from the group consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The method according to any one of claims 20 to 27 , wherein the expression level of the SLFN11 gene at the mRNA level is judged to be high when the log2[FPKM+1] value is greater than 2.0 or 3.0. The method according to claim 28, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 2.0. The method according to claim 28, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[FPKM+1] value is greater than 3.0. EdgeSeq A was performed on biological samples obtained from human patients diagnosed with cancer.
The method according to claim 1 or 2, wherein the log2[MNC+1] value is measured by assay, and if this value exceeds a specific value, the expression level of the hTROP2 gene and/or the SLFN11 gene at the mRNA level is judged to be high. 32. The method according to claim 31, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than any one selected from the group consisting of 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, and 15.0. The method according to claim 31 or 32, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than any one selected from the group consisting of 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, and 14.0. The method according to any one of claims 31 to 33 , wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 12.0, 13.0, or 14.0. The method according to claim 34, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 12.0. The method according to claim 34, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 13.0. The method according to claim 34, wherein the expression level of the hTROP2 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 14.0. The method according to any one of claims 31 to 37, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than any one selected from the group consisting of 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, and 13.5. The method according to any one of claims 31 to 38, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than any one selected from the group consisting of 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, and 12.5. The method according to any one of claims 31 to 39, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[MNC+1] value exceeds any one selected from the group consisting of 11.5, 12.0, and 12.5 . SLFN1 at the mRNA level when the log2[MNC+1] value is greater than 11.5
The method according to claim 40, wherein the expression level of one gene is determined to be high. The method according to claim 40, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 12.0. The method according to claim 40, wherein the expression level of the SLFN11 gene at the mRNA level is determined to be high when the log2[MNC+1] value is greater than 12.5. The method of any one of claims 1 to 43, wherein the biological sample comprises a tumor sample. The method according to any one of claims 1 to 44, wherein the pharmaceutical containing an anti-hTROP2 antibody is an anti-hTROP2 antibody-drug conjugate. The anti-hTROP2 antibody-drug conjugate has the formula

(In the formula, A represents the binding site to the anti-hTROP2 antibody.)
The method according to claim 45, wherein the drug linker represented by the formula: The method according to claim 46, wherein the anti-hTROP2 antibody is an antibody having a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 2. The method of claim 47, wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-hTROP2 antibody is deleted. The method according to any one of claims 46 to 48, wherein the average number of drug-linker structures bound per antibody is in the range of 2 to 8. The method according to any one of claims 46 to 49, wherein the average number of drug-linker structures bound per antibody is in the range of 3.5 to 4.5. The method according to claim 45, wherein the anti-hTROP2 antibody-drug conjugate is Sacituzumab Govitecan (IMMU-132). The method of any one of claims 1 to 51, wherein the cancer is lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, biliary tract cancer, thyroid cancer, lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, and/or multiple myeloma.

Images